Reaction products of methyloxirane with formaldehyde, oligomeric reaction products with aniline and reaction products of methyloxirane with 2,2'-oxydiethanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-03-05 to 2009-04-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Oligomeric reaction product of formaldehyde, aniline and 2-methyloxirane

Molecular formula:

(C6 H6 N)(C7 H8 N)(1 + m)(C3 H6 O)n with m average = 0.37 and n average > 4 and < 11

IUPAC Name:

Oligomeric reaction product of formaldehyde, aniline and 2-methyloxirane

Constituent 2

Reference substance name:

2,2'-Oxydiethanol, propoxylated

Molecular formula:

C4H10O3(C3H6O)n with >=90% n < 9

IUPAC Name:

2,2'-Oxydiethanol, propoxylated

Constituent 3

Reference substance name:

Propane-1,2-diol, propoxylated

Molecular formula:

C3H8O2(C3H6O)n with >=90% n<8

IUPAC Name:

Propane-1,2-diol, propoxylated

Constituent 4

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogenoxide

Test material form:

liquid

Details on test material:

- Trade name: Daltolac R 159
- CAS/NLP number: 25084-89-3/500-031-4
- Batch Sample Ref RZW 0000023
- appearance: yellow liquid
- viscosity at 80°C: 126 mPa
- stored at ambient temperature, in the container in which it was received until required for testing

Method

Target gene:

Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision repair system. Tester strains TA98 and TA100 also contain the pKM101 plasmid (carrying the R factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch repair process.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both fr meshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).

The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox et al., 1990). Tester strain WP2 uvrA has a deletion in the uvrA gene resulting in a deficient DNA excision repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base pair substitution mutations (Green and Muriel, 1976).

Test concentrations with justification for top dose:

The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells.

Details on test system and experimental conditions:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100,
TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as
described by Green and Muriel (1976). Salmonella tester strains were received from Dr. Bruce
Ames’ designated distributor, Discovery Partners International, San Diego, California. E. coli
tester strains were received from the National Collection of Industrial and Marine Bacteria,
Aberdeen, Scotland.

Overnight cultures were prepared by inoculating from the appropriate master plate or from the
appropriate frozen permanent stock into a vessel containing ~50 mL of culture medium. To
assure that cultures were harvested in late log phase, the length of incubation was controlled and
monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room
temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm
at 37±2°C approximately 12 hours before the anticipated time of harvest. Each culture was
monitored spectrophotometrically for turbidity and was harvested at a percent transmittance
yielding a titer of greater than or equal to 0.3x10e9 cells per milliliter. The actual titers were
determined by viable count assays on nutrient agar plates.

Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was
prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of
Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The lot of S9 was prepared by and
purchased from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C
or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize at
least two promutagens to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM
glucose-6-phosphate, 4 mM ß-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and
33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix),
containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To
BioReliance confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective
agar.

Solubility Test
A solubility test was conducted using dimethyl sulfoxide (DMSO) to determine the highest
soluble or workable stock concentration, up to 500 mg/mL.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate
were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean
revertants per plate of at least one tester strain over a minimum of two increasing concentrations
of test article.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean
revertants at the peak of the dose response was equal to or greater than 3.0-times the mean
vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged
positive if the increase in mean revertants at the peak of the dose response was equal to or
greater than 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets
the criteria for evaluation as positive. This could be a dose-responsive increase that does not
achieve the respective threshold cited above or a non-dose responsive increase that is equal to or
greater than the respective threshold cited. A response will be evaluated as negative, if it is
neither positive nor equivocal.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

Historical Negative and Positive Control Values
2005 – 2007

revertants per plate
Strain Control Activation
None Rat Liver
Mean SD Min Max Mean SD Min Max
TA98 Neg 17 6 5 56 25 8 6 60
Pos 189 112 30 1812 526 343 73 2669
TA100 Neg 133 30 52 250 143 32 67 263
Pos 548 148 112 4349 698 343 232 2652
TA1535 Neg 20 7 4 53 16 5 4 45
Pos 420 146 54 1019 104 59 18 985
TA1537 Neg 7 3 1 20 8 3 1 29
Pos 740 356 14 2514 88 87 13 1297
WP2 uvrA Neg 21 9 5 69 23 10 4 65
Pos 214 137 28 1086 268 174 29 1178
SD=standard deviation; Min=minimum value; Max=maximum value; Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

Any other information on results incl. tables

Bacterial Mutation Assay Summary of Results - Initial Toxicity-Mutation Assay
Test Article Id	:		DADPM/DEG-PO
Study Number	:		AC23JM.503.BTL
Experiment No	:		B1
Average Revertants Per Plate ± Standard Deviation
Activation Condition		:		None
Dose (µg per plate)		TA98						TA100						TA1535						TA1537						WP2uvrA
Vehicle		16		±		4		215		±		23		18		±		4		6		±		3		29		±			1
1.5		14		±		4		207		±		5		19		±		4		6		±		1		27		±			2
5.0		14		±		3		196		±		24		17		±		1		3		±		1		33		±			6
15		9		±		3		185		±		27		16		±		0		8		±		0		31		±			6
50		15		±		4		154		±		23		17		±		1		9		±		1		31		±			0
150		9		±		0		146		±		1		19		±		2		6		±		2		31		±			4
500		11		±		1		167		±		1		15		±		0		5		±		1		27		±			1
1500		19		±		1		153		±		3		12		±		1		9		±		1		22		±			1
5000		10		±		1		145		±		6		19		±		1		9		±		2		33		±			5
Positive		278		±		53		651		±		33		489		±		28		1977		±		23		405		±			28
Activation Condition		:		Rat Liver S9
Dose (µg per plate)		TA98						TA100						TA1535						TA1537						WP2uvrA
Vehicle		26		±		1		168		±		23		14		±		1		8		±		0		32		±			8
1.5		29		±		1		160		±		7		17		±		5		11		±		0		31		±			6
5.0		30		±		1		174		±		6		18		±		2		5		±		1		34		±			6
15		29		±		3		180		±		5		14		±		0		5		±		1		28		±			8
50		35		±		3		179		±		3		16		±		1		7		±		4		33		±			1
150		28		±		1		163		±		18		10		±		4		10		±		4		27		±			4
500		41		±		11		164		±		5		17		±		5		8		±		1		37		±			2
1500		29		±		4		188		±		9		16		±		4		6		±		3		36		±			3
5000		31		±		1		163		±		4		13		±		4		8		±		0		37		±			6
Positive		345		±		56		1064		±		67		92		±		3		86		±		1		262		±			25
Vehicle = Vehicle Control
Positive = Positive Control (50 µL plating aliquot)
Plating aliquot = 50 µL

Bacterial Mutation Assay Summary of Results - Confirmatory Mutagenicity Assay
Test Article Id	:		DADPM/DEG-PO
Study Number	:		AC23JM.503.BTL
Experiment No	:		B2
Average Revertants Per Plate ± Standard Deviation
Activation Condition		:		None
Dose (µg per plate)		TA98						TA100						TA1535									TA1537									WP2uvrA
Vehicle		11		±		3		122		±		9		15				±			3		8					±	3			26				±			7
50		17		±		5		107		±		13		17				±			2		5					±	3			34				±			3
150		15		±		6		144		±		16		17				±			8		7					±	3			34				±			6
500		16		±		4		125		±		4		17				±			3		6					±	1			35				±			4
1500		12		±		6		140		±		8		17				±			2		4					±	3			25				±			3
5000		14		±		6		129		±		20		19				±			5		4					±	2			27				±			6
Positive		206		±		24		593		±		81		453				±			12		1674					±	221			352				±			53
Activation Condition		:		Rat Liver S9
Dose (µg per plate)		TA98						TA100						TA1535									TA1537									WP2uvrA
Vehicle		20		±		8		143		±		9		14				±			5		4					±	2			29				±			2
50		19		±		8		119		±		24		12				±			3		6					±	3			29				±			8
150		24		±		2		115		±		3		15				±			1		4					±	3			45				±			6
500		27		±		5		117		±		16		14				±			4		5					±	1			35				±			4
1500		35		±		6		132		±		13		10				±			2		4					±	3			32				±			6
5000		33		±		6		115		±		11		11				±			4		5					±	4			33				±			11
Positive		341		±		49		705		±		51		91				±			14		52					±	12			271				±			45
Vehicle = Vehicle Control
Positive = Positive Control (50 µL plating aliquot)
Plating aliquot = 50 µL

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative under the conditions of this study, test article DADPM/DEG-PO did not cause a positive mutagenic response in either the presence or absence of Aroclor induced rat liver S9.

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, test article DADPM/DEG-PO did not cause a positive mutagenic response in either the presence or absence of Aroclor induced rat liver S9.

Executive summary:

The purpose of this study was to evaluate the mutagenic potential of the test article by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9.

The test article, DADPM/DEG-PO, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. After vortexing for 10 minutes and sonication at 18°C for 2 minutes, the test article formed a soluble and clear solution at approximately 500 mg/mL, the maximum concentration tested.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. In the initial toxicity-mutation assay, no positive mutagenic response was observed. Precipitate was observed at 5000 µg per plate. No appreciable toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 µg per plate.

In the confirmatory mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate. Precipitate was observed at 5000 µg per plate with a few test conditions. No appreciable toxicity was observed. 

Under the conditions of this study, test article Daltolac XR 159 (DADPM/DEG-PO) was concluded to be negative in the Bacterial Reverse Mutation Assay.